Rett syndrome is a neurodevelopmental disorder typically due to mutations in the methyl CpG binding protein, MeCP2. An aspect of MeCP2 biology that has received relatively little attention is its regulation at the level of translation. The long isoform of the MeCP2 transcript (the predominant transcript found in brain) contains blocks of sequence that are highly conserved evolutionary, including some regions that have not undergone a single nucleotide change over tens of millions of years. We propose that these sequences are binding sites for a family of microRNAs whose expression is driven by CREB, a critical signal-dependent transcriptional activator. Our overall hypothesis is that CREB directs the expression of a family of microRNAs that regulate MeCP2 translation. The ability of CREB to stimulate microRNA expression is, in turn, under the control of REST, a well-known represser of neural genes. This model provides a mechanism for CREB (as well as the pathways that activate CREB function) to regulate MeCP2 protein levels over the long term. This mechanism is proposed to prevent the deleterious effects of increased MeCP2 protein levels that may occur after certain types of neuronal activity. Our specific aims are to 1) Determine whether five CREB-regulated microRNAs, namely miR132, miR191, miR320, miR219-1, and miR328 inhibit MeCP2 protein expression. Specific 2'-O-methyl oligonucleotides will be used singly and in combination to determine whether they block the microRNA-mediated changes in MeCP2 levels. The effects of the microRNAs on the binding of MeCP2 to the BDNF promoter will also be determined;-2) Characterize the developmental and signaling pathways that regulate expression of the five microRNAs;3) Determine whether the developmental regulation of MeCP2 by miR132 is controlled by REST. There is virutally nothing known about the transcriptional regulation of microRNAs or the factors that control MeCP2 protein expression. The studies described in this proposal address a completely novel mechanism of gene regulation that has implications for the understanding of MeCP2 biology and neural development in general.